tbuckley/cabot-search huggingface dataset:
Abstract P1032: Substrate Optimization For Patients With Melas And Leigh Syndrome Using Induced Pluripotent Stem Cells-derived Cardiomyocytes
Authors: Taylor H Yamamoto, Constance C Huang, Ava K Cutright, Kenan C Dinchman, Bruce H Cohen, William M Chilian, Patrick T Kang
Journal: Circulation Research (2023)

Mitochondrial DNA (mtDNA) mutations increase the risk of health issues with overlapping clinical phenotypes. We propose that patient-derived induced pluripotent stem cells (iPSCs) can serve as surrogates for studying mitochondrial diseases, provided a standardized protocol is used to mitigate effects of mtDNA heteroplasmy. Accordingly, we reprogrammed peripheral blood mononuclear cells (CytoTune Kit) from mtDNA disease patients with epilepsy enrolled in clinical trials. After culturing the reprogrammed cells, six iPSC cell colonies were selected (Alkaline Phosphatase Live Stain) from each donor, pooled, and sorted by SSEA4+ marker at passage 6. Pluripotency was confirmed using OCT4 immuno-staining and RT-PCR for OCT4, NANOG, and SOX2 at passage 10. Because two of the patients (m.3243A&gt;G MT-TL1, MELAS, and m.T8993G MT-ATP6, Leigh Syndrome) showed signs of improvement on drug therapy, we differentiated their iPSCs into cardiomyocytes (iCMs) and characterized them using immuno-staining (cTnT). We then screened the substrate utilization preferences of the iCMs using Phenotype MicroArray Mammalian assays (Biolog). The MELAS-iCMs failed to metabolize lactic acid, L-ornithine, and branched-chain amino acids, but performed better than control iCMs in the presence of arginine and glycerol phosphate. In contrast, LS-iCMs exhibited distinctly different substrate preferences and thrived in the presence of acetoacetic acid and α-keto-butyric acid with reduced oxidative stress (AmplexRed). The failure of MELAS-iCMs to survive in the presence of only lactic acid recapitulates a characteristic feature of MELAS, underscoring the potential of iPSCs for disease modeling. Our findings suggest that diverse outcomes to nutritional and pharmaceutical interventions must be tailored to each patient. To validate the pharmacological efficacy of iCMs, we will continuously evaluate their response in patients who are refractory to treatment.
Abstract P1052: Development Of A Porcine Model Of Arteriovenous Fistula For The Evaluation Of Radiopaque Polymeric Vascular Wrap
Authors: Carleigh Klusman, Benjamin Martin, Joy Perez, Huckie Del Mundoa, Jossana Damasco, Erin Marie San Valentin, Marvin Bernardino, Allan Barcena, Malea Williams, Steve Parrish, William Norton, Gouthami Chintalapani, Natalie Fowlkes, Tam Huynh, Steven Huang, Marites P Melancon
Journal: Circulation Research (2023)

Purpose: The success and clinical utility of arteriovenous fistulas (AVF) are complicated by neointimal hyperplasia (NIH). Radiopaque and drug-eluting perivascular wraps have demonstrated attenuation of NIH in a rat model, but these constructs have yet to be tested in a large animal. This study aims to create an AVF model in pigs to evaluate the radiopacity and patency of a novel absorbable medical device made of rosuvastatin (ROSU) and gold nanoparticle (AuNP)-loaded polycaprolactone (PCL). Materials and Methods: A solution containing PCL, AuNP, and ROSU was electrospun into 6 x 5 x 0.2 cm scaffolds, and physicochemical properties were characterized over 8 weeks. AVFs with and without implantation of PCL wrap were surgically created in the neck and groin of 3 pigs. In vivo ultrasound (US) and computed tomography (CT) imaging were obtained at weeks 2 and 4. Results: PCL and PCL-Au-ROSU polymeric wraps were successfully fabricated using an electrospinning system. Figure 1 demonstrates successful creation of AVFs in pigs. In vivo Doppler US of AVF at weeks 2 and 4 with or without perivascular wrap showed arterial-venous communication with high-velocity flow and arterialized waveforms in draining veins. CT imaging demonstrated persistent radiopacity of PCL-Au-ROSU wraps through week 4. Conclusion: Outcomes suggest that surgical implantation of PCL wraps during AVF creation is feasible in a pig model. The increased radiopacity seen with AuNPs supports the efficacy of embedded metallic particles for device visualization. Ongoing in vivo studies will focus on optimization of the polymeric perivascular wrap and the evaluation of its efficacy in maintaining AVF patency.
Abstract P1038: Role Of Hypomorphic Notch1 Mutations In Cardiomyocyte Signaling And Function In Hypoplastic Left Heart Syndrome
Authors: Anita Saraf, Schlegel Brent, Javed Zaineb, Krithika Rao, Travis Lear, Jagannathan Rajaganapathi, Mousumi Moulik, Hempel Nadine, Sruti Shiva, Dhivyaa Rajasundaram, Bernhard Kuhn, Toren Finkel
Journal: Circulation Research (2023)

Background: Congenital heart disease (CHD) affects is the most common birth defect and affects 1% of births in the United States. Hypoplastic left heart syndrome (HLHS) a complex CHD where left sided structures are malformed during embryonic development. Compound heterozygous mutations in NOTCH1, a developmental transmembrane protein have been implicated HLHS patients. However, the effect of hypomorphic NOTCH1 expression on cardiomyocyte development has not been fully characterized. Methods: Using HLHS patient derived induced pluripotent stem cells (iPSCs) with a compound heterozygous NOTCH1 expression and CRISPR-Cas9 gene editing technology we developed multiple iPSCs clones with hypomorphic NOTCH1 expression. We characterized expression of NOTCH1 downstream signaling molecules using transcriptomics of iPSCs and CMs. We further characterized mitochondrial structure, ATP production and sarcomere architecture of the resulting CMs. Lastly, we characterized calcium transients and contractility of the NOTCH1 hypomorphic CMs. Results: iPSC-CMs with hypomorphic NOTCH1 expression showed reduced pluripotency and decreased differentiation into cardiomyocytes. Transcriptomics studies showed that hypomorphic clones increasingly differentiated into fibroblasts and smooth muscle cells as compared to wild-type (WT) cells. Multiple signaling pathways associated mitochondrial function, sarcomeric organization, and cardiomyocyte development were dysregulated. Live cell imaging showed abnormal sarcomeric architecture and mitochondrial hyperfission as compared to WT cells. ATP production was also decreased as compared to WT. Lastly, calcium transients in hypomorphic CMs showed a decrease in time to peak and upslope which consequently lead to decreased contractility and relaxation rates of hypomorphic NOTCH1 CMs. Conclusion: Hypomorphic expression of NOTCH1 seen in HLHS affects multiple aspects of CM cytoskeletal architecture during CM development. Consequently, NOTCH1 hypomorphic iPSC-CMs have abnormal calcium transients and decreased contractility, likely contributing to the higher incidence of arrhythmia and heart failure seen in HLHS patients.
Abstract P2120: Alteration Of Left Ventricular Load Impacts Exosomal And Cardiac MicroRNA Expression In A Porcine Model Of Myocardial Ischemia
Authors: Jonas M Marx, Renata Mazurek, Olympia Bikou, Tomoki Sakata, Spyros M Mavropoulos, Susmita Sahoo, Kiyotake Ishikawa
Journal: Circulation Research (2023)

Background: Mechanical unloading of the left ventricle (LV) after myocardial infarction (MI) exhibits cardioprotective effects. The underlying molecular mechanisms of these benefits are not fully elucidated. MicroRNAs (miRNA) are vital regulators of cellular processes. When released into circulation within exosomes, they participate in inter and intra-organ communications. We hypothesized that LV load influences exosomal and myocardial miRNA expression. Methods: Anterior MI was induced in Yorkshire pigs. After one week, pigs underwent mechanical LV unloading per Impella or LV overloading by percutaneous aortic valve destruction (n = 5 each). Two hours later, coronary sinus blood was collected, animals were euthanized and infarct border zone tissue was harvested. Exosomal miRNAs isolated from blood were studied using next-generation sequencing. Myocardial tissue expression of select differentially regulated exosomal miRNAs was assessed by quantitative PCR. Results: In coronary sinus blood from LV unloaded animals exosomal miR-19b, miR-20a, miR-21, miR-144, and miR-146b showed higher expression compared to LV overloaded pigs (log2 FC = 3.47, 2.64, 1.86, 2.62, 1.58; p&lt;0.05). Opposite relationships were observed in myocardial tissue. MiR-19b and miR-20a showed lower expression in LV unloaded compared to overloaded animals (FC = 0.36 ± 0.11 and 0.37 ± 0.12, p&lt;0.05). A similar trend was found for miR-21, miR-144, and miR-146b expression. Conclusion: Exosomal miRNAs in coronary sinus blood are affected by changes in LV load. Inverse expression patterns in myocardial tissue suggest increased cardiac release or decreased uptake of exosomes as a possible mechanism.
Abstract P1184: Spatiotemporal Proteomics Approach To Discern New Features Of Carfilzomib-mediated Cardiotoxicity
Authors: Jordan Currie, Vyshnavi Manda, Veronica Hidalgo, Robert W Ludwig, Maggie P Lam, Edward Lau
Journal: Circulation Research (2023)

Background: Proteasome inhibitors including carfilzomib have led to remarkable improvement in the survival of multiple myeloma patients, but are associated with cardiovascular adverse events including heart failure. A better understanding of the mechanism of toxicity is needed to improve oncogenic and cardiovascular outcomes. Rationale: Proteasome inhibition is known to impair the degradation rates as well as the subcellular distribution of proteins, processes that are not fully reflected by differential gene expression analysis. We hypothesize that cardiomyocyte proteins show impaired spatiotemporal distributions upon carfilzomib exposure, and that these changes constitute a crucial under-examined aspect of cardiotoxicity mechanism. Approach: To test this hypothesis, we developed a new mass spectrometry-based proteomics strategy that can simultaneously trace the turnover kinetics and subcellular localizations of proteins in cardiac cells. Results: In preliminary work, we applied this method to human iPSC-derived cardiomyocytes under 0.5 μM carfilzomib vs. control (n=2). We quantified the localization and turnover rates of &gt;3,500 proteins in control and carfilzomib-treated cells. Unexpectedly, no significant difference was identified in the global rate of protein turnover between control and carfilzomib treated cells (p = 0.24 Wilcoxon-Mann-Whitney test), where the median protein half-lives of of control and carfilzomib treated cells were 31.0 vs. 30.7 hours, respectively. However, when we used a T-augmented Gaussian mixture model to predict protein localizations and assess compartment-specific turnover changes, we observed a significant reduction in turnover in the nuclear fraction, and a concomitant increase in that of proteasomal fraction under carfilzomib. The data also revealed a change in assigned localization in many myosin-binding proteins toward lysosomal co-sedimenting compartments, consistent with sarcomeric disarray and degradation in carfilzomib-exposed cardiomyocytes. Finally, we show a decrease in turnover of both fatty acid and glucose metabolism associated proteins. This method has potential for discovering unique molecular features of proteasome inhibitor mediated cardiotoxicity.
Abstract P1143: Med12 “Mediates” Transcriptional Regulation In The Heart
Authors: Dominic Kolonay, Kedryn K Baskin
Journal: Circulation Research (2023)

The Mediator complex serves as a bridge to link transcriptional machinery and transcription factors (TFs) to control transcription, but the molecular mechanisms of transcriptional regulation by Mediator are not well-understood. Mediator is a large multiprotein complex organized as submodules, known as the head, body, tail, and kinase submodule. The CDK8 kinase submodule is known to regulate Mediator - RNA Polymerase II interaction. MED12 is an essential Mediator component, and within the kinase submodule, is required for CDK8 kinase activity. MED12 protein levels are increased in human heart failure biopsies and in the mouse heart after transverse aortic constriction (TAC)-induced heart failure. To test the hypothesis that increased levels of MED12 contributes to development of heart failure we generated cardiac-specific Med12 transgenic mice (cTg) by driving increased MED12 expression in cardiomyocytes using the αMHC promoter. Med12 overexpression leads to decreased cardiac function, measured as fractional shortening by serial echocardiography. Decreased cardiac function was accompanied by increased cardiac chamber size and dilation, without cardiac fibrosis. RNA sequencing of cardiomyocytes from cTg and control mice revealed that increased levels of MED12 leads to dysregulated calcium handling gene expression. MED12 is a transcriptional regulator, but does not directly bind DNA. To determine how MED12 regulates gene expression we screened for TFs that interact with MED12. We performed immunoprecipitation of MED12 and by mass spectrometry and determined that MED12 interacts with the TFs MEF2, CREB, and SRF. In follow-up experiments we demonstrated that MED12 interacts with the transcription factor (TF) MEF2 to regulate calcium handling genes. Current studies are focused on determining how MED12 binding coordinates gene expression through multiple TFs in cardiomyocytes. Collectively, our data demonstrate that increased MED12 expression leads to the development of heart failure, in part by dysregulation of gene expression. Therefore, precise regulation of MED12 levels is important to maintain normal cardiac function.
Abstract P1123: Phf7 As A Critical Driver Of Adult Cardiac Reprogramming
Authors: Glynnis A Garry, Kenian Chen, Huanyu Zhou, Hisayuki Hashimoto, Svetlana Bezprozvannaya, Ning Liu, Rhonda S Bassel-Duby, Eric N Olson
Journal: Circulation Research (2023)

Direct cardiac reprogramming of fibroblasts to cardiomyocytes presents an attractive therapeutic strategy to restore cardiac function following injury. Cardiac reprogramming was initially achieved through the overexpression of the transcription factors Gata4, Mef2c, and Tbx5 (GMT), and later, Hand2 (GHMT) and Akt1 (AGHMT) were found to further enhance this process. Yet, staunch epigenetic barriers severely limit the ability of these cocktails to reprogram adult fibroblasts. We undertook a screen of mammalian gene regulatory factors to discover novel regulators of cardiac reprogramming in adult fibroblasts and identified the histone reader PHF7 as the most potent activating factor. Mechanistically, PHF7 localizes to cardiac super enhancers in fibroblasts, and through cooperation with the SWI/SNF complex, increases chromatin accessibility and transcription factor binding at these sites. Further, PHF7 recruits cardiac transcription factors to activate a positive transcriptional autoregulatory circuit in reprogramming. Importantly, PHF7 is the first epigenetic factor shown to achieve efficient reprogramming in the absence of Gata4. Here, we highlight the underexplored necessity of cardiac epigenetic modifiers, such as PHF7, in harnessing chromatin remodeling and transcriptional complexes to overcome critical barriers to direct cardiac reprogramming.
Abstract P1120: Chromobox 7 Represses Cardiomyocyte Proliferation, And Its Genetic Ablation Promotes Cardiac Regeneration Following Myocardial Injury
Authors: Kyuwon Cho, Mark Andrade, Seongho Bae, Sangsung Kim, Jin Eyun Kim, Er Yearn Jang, Sangho Lee, Ahsan Husain, Roy L Sutliff, John W Calvert, Changwon Park, Young-sup Yoon
Journal: Circulation Research (2023)

Background: Cardiomyocyte (CM) proliferation decreases after birth but the underlying mechanism is poorly understood. Chromobox 7 (CBX7) regulates the cell cycle but its role in CM proliferation is unknown. Methods: We profiled CBX7 expression in the mouse hearts via qRT-PCR, western blotting, and immunohistochemistry. We knocked down CBX7 by using constitutive and inducible conditional knockout mice (Tnnt2-cre;Cbx7 fl/+ and Myh6-MCM;Cbx7 fl/fl , respectively). We then measured CM proliferation by immunostaining. To examine the role of CBX7 in cardiac regeneration, we employed neonatal cardiac apical resection and adult myocardial infarction (MI) models. We examined the mechanism of CBX7-mediated repression of CM proliferation via co-immunoprecipitation, mass spectrometry, and other molecular techniques. Results: The mRNA expression of Cbx7 was increased at the perinatal stage and sustained in the postnatal heart. The haplodeficiency of Cbx7 (Tnnt2-Cre;Cbx7 fl/+ ) promoted proliferation of neonatal CMs in vivo , leading to increased myocardial wall thickness. Genetic deletion of Cbx7 in CMs ( Cbx7 iCKO, Myh6-MCM;Cbx7 fl/fl ) at P0 resulted in increased CM proliferation (% of Ki67 + CMs: vehicle, 9.5 vs. tamoxifen, 18.3, P &lt; 0.01). In response to cardiac apical resection surgery at P7, Cbx7 iCKO mice exhibited better cardiac function (% of LVEF: vehicle, 44.1 vs. tamoxifen, 54.7, P &lt; 0.05) and less fibrosis at P28 compared to the control. Following the MI surgery, adult (5-months-old) Cbx7 iCKO mice exhibited better cardiac function (% of LVEF: vehicle, 41.4 vs. tamoxifen, 51.5, P &lt; 0.05) and less fibrosis at one month post-MI compared to the control. Mechanistically, CBX7 interacted with TARDBP and positively regulated its downstream target, RBM38, an inducer of the cell cycle arrest, in a TARDBP-dependent manner. Rbm38 was perinatally upregulated in the mouse hearts and overexpression of Rbm38 reduced proliferation of neonatal CMs. Conclusions: CBX7 expression is perinatally increased and inhibits proliferation of CMs by controlling TARDBP/Rbm38 pathway. Genetic ablation of Cbx7 promoted regeneration of neonatal and adult hearts. This is the first study to uncover the role of CBX7 in regulation of CM proliferation and cardiac regeneration.
Abstract P1019: High-throughput Generation And Functional Characterization Of Myosin Heavy Chain 7 Variants In Genome-edited Cardiomyocytes
Authors: Kai-Chun Yang, Clayton Friedman, Shawn Fayer, Sriram Pendyala, Wei-Ming Chien, Alexander Loiben, Leslie Chao, Dania Ahmed, Elaheh Karbassi, Charles Murry, Lea Starita, Douglas Fowler
Journal: Circulation Research (2023)

Hypertrophic cardiomyopathy (HCM), an inheritable cardiac condition characterized by idiopathic left ventricular hypertrophy, can cause sudden cardiac death or heart failure. Autosomal-dominant missense mutations in MYH7 (encoding the sarcomeric βMHC protein) account for 33% of genotype-positive HCM cases. While pathogenic MYH7 variants are clinically-actionable, 75% of MYH7 missense variants in ClinVar are variants of unknown significance (VUS). Gene-editing human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) can help determine the function of MYH7 VUS, however, the discovery of new variants by genetic testing outpaces the ability to gene-edit hiPSCs. Thus, we developed a novel method called CR ISPR a On- T arget E diting R etrieval (CRaTER) which increases the success of gene-editing by 25-fold compared to current methods. We leveraged CRaTER to enrich for hiPSCs edited with a library of MYH7 missense variants and recovered 113 different heterozygous variants. As deleterious mutations can reduce protein stability, we flow sorted MYH7 variant hiPSC-CMs into bins based on differing βMHC abundance followed by next generation sequencing of each bin to calculate variant abundance scores. This assay identified 31 MYH7 missense variants as functionally abnormal (lower abundance scores than synonymous controls), including all known pathogenic variants in the library. This βMHC depletion phenotype was validated in clonal hiPSC-CMs and supported by western blotting of myocardium from a patient with a pathogenic MYH7 missense mutation. Furthermore, we rescued βMHC protein levels by genetically ablating the gene encoding for Muscle RING-finger protein-1, an E3 protein ligase that targets βMHC for degradation, and found that the cardiomyocyte hypertrophy induced by the pathogenic MYH7 E848G HCM variant was significantly blunted. In summary, CRaTER enables the generation of gene-edited hiPSC lines at unprecedented scale. Functional analysis of MYH7 variant hiPSC-CMs reveals a novel correlation between loss of βMHC protein and clinical pathogenicity that can be leveraged for clinical variant interpretation. Interventions that prevent the loss of βMHC may potentially be therapeutic for MYH7 -associated HCM.
Abstract P1029: Abnormal Progenitor Cell Differentiation And Cardiomyocyte Proliferation In Hypoplastic Right Heart Syndrome
Authors: Mingtao Zhao, Yang Yu, Shiqiao Ye, Vidu Garg, zhaohui xu
Journal: Circulation Research (2023)

Pulmonary atresia with intact ventricular septum (PA-IVS) is a rare (4-8 per 100,000 live births) type of hypoplastic right heart syndrome (HRHS) where the right-sided structures in the heart are malformed. In PA-IVS, the pulmonary valve that acts to regulate the unidirectional flow of blood from the right ventricle (RV) to the lungs does not open, resulting in no connection between the RV and the pulmonary arteries. After surgical or catheter-based intervention, PA-IVS patients have clinical outcomes that range from single-ventricle palliation to a biventricular repair. Mechanisms underlying the spectrum of RV hypoplasia in PA-IVS are difficult to fully ascribe to an atretic pulmonary valve. There are no reliable animal models available for studying disease mechanisms of PA-IVS. In this study, we leverage patient-specific induced pluripotent stem cells (iPSCs) and single-cell RNA sequencing to elucidate cellular etiologies of ventricular hypoplasia in PA-IVS. PA-IVS iPSC-derived cardiomyocytes are less proliferative compared to controls under both static and cyclic stretch, suggesting that genetic factors may contribute to a spectrum of ventricular hypoplasia in PA-IVS. Single-cell transcriptomic analysis reveals that cell lineage determination towards second heart progenitors is suppressed, with enhanced differentiation into first heart field and epicardial lineages during cardiac differentiation. Additionally, biological pathways associated with cell proliferation and cell cycle progression are downregulated whereas mitochondrial activities are elevated in early cardiomyocytes originated from PA-IVS patients. In conclusion, abnormal cell lineage differentiation and cardiomyocyte proliferation may underlie the cellular and developmental etiologies of ventricular hypoplasia in HRHS.